The research of the lab has demonstrated that voltage-dependent and ligand-activated channels are expressed during early differentiation of amphibian spinal neurons in vivo and develop with a similar schedule in dissociated cell culture. Further, channels appear in sequential order and their properties are modified as development proceeds. These findings suggest that ion channel activity can participate in signal transduction that influences subsequent steps of development. In fact, our recent work indicates that spontaneous electrical activity and associated calcium influx affect later aspects of differentiation, in vitro. The proposed studies address the roles that rapidly signalling ion channels play in slower molecular events involved in regulation of neuronal differentiation, in vivo. There are three specific aims. Our first aim is to analyze spontaneous elevations of intracellular calcium ([Ca2+]i) occurring in vivo. Calcium indicator fluorescence in the isolated spinal cord (confocal imaging) will be used to measure changes in [Ca2+]i and determine their developmental stage and positional dependence. This activity will be classified as random, clustered or propagated. The mechanisms leading to initiation and subsequent elevation of [Ca2+]i will be analyzed initially by determining the roles of metabolites, growth factors, and transmitters in modulating calcium and potassium currents that my trigger spontaneous activity (imaging & whole cell voltage-clamp). Our second aim is to analyze the molecular basis of action [Ca2+]i in driving calcium-dependent differentiation. Recent data demonstrating elevated intranuclear calcium motivate investigation of transcriptional responses to elevation of [Ca2+]i in embryos injected with calcium-dependent transcription elements in tandem with a reporter. In parallel, activation of endogenous calcium-regulated genes by calcium elevation will be analyzed, and age-dependent changes in their activation will be studied (RNAase protection, northerns and in situ hybridization). Our third aim is to define the responses to GABA and its consequent effects on [Ca2+]i resulting from activation of low threshold calcium currents. Several different varieties of GABA receptors of Rohon-Beard sensory neurons are being characterized in vivo. The functional significance of each class during development will be assessed at different stages by determining the effects of GABA receptor activation on [Ca2+]i in vivo (whole cell voltage-clamp & imaging) and on neuronal survival, neurite outgrowth as well as transmitter sensitivity in vitro. The immediate goal is to test hypotheses about specific mechanisms underlying differentiation of vertebrate spinal neurons in order to define the role of early ion channel activity in driving differentiation. The long term goal is to provide information about the cellular and molecular machinery that governs processes of development. It is expected that this work will promote understanding of developmental disorders of the central nervous system.